CN114660053B - Preparation and application of microfluid aggregation-based fish flesh freshness detection paper-based chip - Google Patents
Preparation and application of microfluid aggregation-based fish flesh freshness detection paper-based chip Download PDFInfo
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Abstract
The invention provides a method for manufacturing and detecting a paper-based chip for detecting freshness of fish meat based on microfluid aggregation. Xanthine Oxidase (XOD) and horseradish peroxidase (HRP) are fixed on a nitrocellulose membrane, 3 ', 5, 5' -Tetramethylbenzidine (TMB) is used as a color developing agent in parallel to prepare a paper-based chip, and the detection of hypoxanthine in fish is realized. The paper-based chip is directly attached to the surface of an aquatic product, the base liquid on the surface of fish can form microfluid through different apertures and gather to the position of immobilized enzyme, so that the direct detection of hypoxanthine is realized, and interference matrix components such as protein and the like can be intercepted due to the difference of the apertures of polysaccharide modification layers with different molecular weights, so that the interference of the matrix is reduced, and the direct naked-eye detection of the freshness of the aquatic product is realized.
Description
Technical Field
The invention relates to the technical field of paper-based detection, in particular to manufacture and application of a microfluid aggregation-based fish flesh freshness detection paper-based chip.
Background
Fish meat is the most digestible of all meat species, and the conversion rate of digestion and subsequent absorption by the human body can reach 90%. The fish meat has high protein content and is easy to be digested and absorbed by human body. The fish meat also contains different kinds of amino acids required by human bodies, wherein leucine and lysine are two large amino acids which are rich in fish with the most requirements of the human bodies. The fat content in the fish meat is relatively low, and is only 1% -10%, which is greatly lower than that of livestock meat. The fat in the fish meat is also easy to be digested and absorbed by people, the fish meat is neutral, and the digestibility of people is close to one hundred percent. The fish meat has rich inorganic salt content and variety, which is ten to fifty times more than that of animal meat and also over ten kinds. In addition, the fish meat also contains a large amount of trace elements essential to human body. Therefore, the fish meat has a very high nutritional value.
The detection of the freshness of the fish is related to various aspects of safety, sanitation, nutrition degree and the like of the fish product, and is very important in the processing, production, circulation and consumption processes of the fish product. The selection of a proper marker has important significance for the quality evaluation of aquatic products. Hypoxanthine, an important alkaloid, is the major catabolite of Adenosine Triphosphate (ATP). After the aquatic product dies, ATP in the body generates a series of degradation reactions under the action of enzyme to generate hypoxanthine. Compared with other characteristic markers of the freshness of aquatic products, the estramustine is generated in the early deterioration stage of the aquatic products. Therefore, accurate and rapid determination of the hypoxanthine content is beneficial to assessment of the early freshness of aquatic products, and has important significance for better assessment of the quality of aquatic products in the transportation process and further adjustment of transportation environments and the like.
At present, the mode of evaluating the freshness of aquatic products mainly comprises a traditional sensory evaluation method and an instrument detection method. The subjective evaluation of the sense is strong, and only the rotten aquatic products can be judged. The instrument detection method mainly comprises methods such as high performance liquid chromatography, capillary column gas chromatography, capillary electrophoresis, fluorescence mass spectrometry, electrochemistry and the like. These methods also have many limitations, such as complicated operation, long detection time, expensive instruments and professional operation. In addition, the complex matrix composition in fish meat causes the detection of freshness to often require a more complex pretreatment process. The pretreatment of the sample, including tissue trituration, high-speed centrifugation and repeated extraction and purification, not only needs relatively complete experimental equipment, but also generally consumes more than 2-3 hours or even longer time. Therefore, in the past, some methods for detecting the freshness of fishes are expensive in experimental instruments and equipment, operation methods need professional workers to perform professional operations, the time and the labor are immeasurable, and the implementation difficulty is high. In order to make up for the defects of the prior art, on one hand, a simple detection method needs to be developed, and the carrying and the application are convenient; on the other hand, the method can overcome the complex matrix interference and remove the interference of macromolecular substances; thirdly, a non-subjective result discrimination method needs to be established, such as a discrimination method based on a smart phone.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a preparation method and application of a microfluid aggregation-based fish flesh freshness detection paper-based chip.
The invention is realized by the following technical scheme: a manufacturing method of a paper-based chip for detecting freshness of fish meat based on microfluid aggregation specifically comprises the following steps:
s1, preparing a required modification liquid M of the paper-based chip:
s11, preparing a color development liquid: weighing 0.096 g of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) powder, dissolving in 1 mL of DMSO to prepare TMB mother liquor with the concentration of 400 mM, and diluting to 60-100mM for later use;
s12, preparing a horseradish peroxidase (HRP) mother solution: HRP was dissolved in phosphate buffer (PBS, 20 mM, pH = 7.4) to make 5000U/mL of stock solution. Continuously diluting to 20-100U/mL for later use;
s13, preparing modification liquid M: mixing a chitosan oligosaccharide lactic acid (COL) solution, a developing solution and a 25U/mL HRP solution according to the ratio of N: 1: 1 (volume ratio, N = 3-8) and uniformly mixing;
s2, preparing a required modification liquid N of the paper-based chip:
s21, preparing a Xanthine Oxidase (XOD) solution: dissolving XOD in phosphate buffer (PBS, 20 mM, pH = 7.4) to prepare 100-500U/mL enzyme solution;
s22, preparing a modifying liquid N: uniformly mixing 300U/mL XOD solution and 1wt% chitosan solution according to an equal volume ratio;
s3, paper base cutting: cutting the paper base material into squares of 1.5 cm by 1.5 cm to be used as paper bases of the chips;
s4, modification in the first step: sucking 0.7 μ L of modifying solution M, dripping onto NC membrane, and drying at room temperature for 2 min;
s5, second-step modification: after drying, 1 mu L of modification liquid N is dripped on the site which is just modified, and drying is carried out for 1.5 min at room temperature, thus finishing modification;
s6, according to the same method of S3-S4, at most 4 detection sites can be modified on a single paper base;
s7, pre-packaging of the paper-based chip: packaging 5 manufactured paper-based chips into 6 cm by 10 cm aluminum foil vacuum packaging bags, and performing vacuum-pumping treatment and hot plastic packaging by using a vacuum-pumping machine;
s8, storage: and (4) storing the packaged paper-based chip in a refrigerator at 4 ℃ for later use.
Preferably, the chitosan oligosaccharide lactic acid (COL) solution in step S13 is prepared by weighing 0.2-0.3 g of COL powder (5-10 kDa), adding 9.8 mL of water and 0.2 mL of acetic acid, and heating at 50 deg.C with stirring for 10min until the chitosan oligosaccharide lactic acid is dissolved.
As a preferable example, the 1wt% chitosan solution in step S22 is prepared by weighing 0.05-0.1 g of chitosan solution powder (100-300 kDa), adding 9.8 mL of water and 0.2 mL of acetic acid, and heating at 50 ℃ with stirring for 10min until the chitosan is dissolved.
Preferably, the paper base material in step S3 includes one of nitrocellulose membrane, Whatman No. 1 chromatography paper, baowerd filter paper made in China, and dyed paper.
Further, the paper base material is a nitrocellulose membrane (NC membrane).
A detection method for detecting a paper-based chip based on microfluid aggregated fish freshness specifically comprises the following steps:
t1, drawing a hypoxanthine standard curve:
t11, hypoxanthine concentration gradient set to 0.01-5.2 mM;
t12, shooting the result, wherein the shooting specification is as follows: selecting a mobile phone photographing mode as a professional mode; the light source is selected as a built-in LED light source of the mobile phone; a sensitivity ISO selection of 50; selecting a light measuring mode as a spot light measuring mode; shutter speed S is set to 1/36; the exposure compensation EV is set to 0; the focusing mode AF is selected as single automatic focusing; the white balance mode WB is set to automatic white balance AWB; when the mobile phone takes a picture, the distance between the mobile phone and the target is 25 cm, and no external light source is arranged; and (3) color intensity modeling treatment: firstly, a picture shot by the mobile phone is transmitted to a computer and opened by using Image J software. And selecting 30 × 30 pixel units in a frame of the part with changed color in the picture by using a selection function, and obtaining the average R, G and B values of the selected area by using a software Histogram function Analyze-Histogram. Processing the obtained RGB value by an Euclidean distance model D, wherein the Euclidean distance model D is represented by a formula (1); the results for color intensity in subsequent experiments were all processed in this way without special mention.
In the formula: r 0 ,G 0 ,B 0 Average RGB values of the blank group detection areas respectively; r i ,G i ,B i Respectively representing the average RGB values of the ith group of experimental color development areas;
drawing the result into a standard curve, and calculating a detection limit and a linear range;
t2, skin detection experiment of fish samples: taking out the sea bass blocks which are placed at 4 ℃ for 6, 5, 4, 3, 2 and 1 days, placing at room temperature for 10min, and then attaching the paper-based chip to the surface of the fish for 3 min, wherein the image acquisition and data processing method is the method of T12;
t3, creation of mobile terminal freshness detection applet: the acquisition of target color is realized by developing a small program function, a color signal is converted into an average RGB value, the numerical value is converted through a formula (1) and corresponds to a standard curve, the freshness of a sample is judged according to the obtained hypoxanthine concentration, and finally, an edible suggestion is given.
Preferably, the linear range of the detection of the target hypoxanthine in the step T12 is 0.01 mM-0.16 mM, the linear regression equation is y =492.722x + 32.798, wherein y is the color intensity, x is the concentration of the hypoxanthine, and the coefficient R is determined 2 0.9903, the detection limit LOD is 8.22. mu.M.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following beneficial effects: the method utilizes polysaccharides with different molecular weights to modify a nitrocellulose membrane layer by layer, and the modified nitrocellulose membrane has a membrane porous structure, a large molecular weight pore structure and a small molecular weight pore structure so as to form a microfluid aggregation effect. Xanthine Oxidase (XOD) and horseradish peroxidase (HRP) are fixed on a nitrocellulose membrane, 3 ', 5, 5' -Tetramethylbenzidine (TMB) is used as a color developing agent in parallel to prepare a paper-based chip, and the detection of hypoxanthine in fish is realized. The paper-based chip is directly attached to the surface of an aquatic product, the base liquid on the surface of fish can form microfluid through different apertures and gather to the position of immobilized enzyme, so that the direct detection of hypoxanthine is realized, and interference matrix components such as protein and the like can be intercepted due to the difference of the apertures of polysaccharide modification layers with different molecular weights, so that the interference of the matrix is reduced, and the direct naked-eye detection of the freshness of the aquatic product is realized.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic diagram of a paper-based chip fabrication process;
FIG. 2 is a graph showing the microfluidic assembly effect exhibited by the prepared paper-based chip, wherein a is the modification method herein; b is Whatman No. 1 chromatographic paper; c is domestic Baoweide filter paper; d is dyed paper;
FIG. 3 is a standard curve (left) and a linear range (right) of detection of hypoxanthine by a paper-based biosensor;
FIG. 4 is a color signal for detecting fish freshness by paper-based chip attachment;
FIG. 5 is a mobile-side freshness fast detection applet.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and thus the scope of the present invention is not limited by the specific embodiments disclosed below.
The following describes the production and application of the microfluid aggregation based fish freshness detection paper-based chip according to the embodiment of the present invention with reference to fig. 1 to 5.
Example 1
As shown in figure 1, the manufacturing method of the paper-based chip for detecting fish freshness based on microfluid aggregation specifically comprises the following steps:
s1, preparing a required modification liquid M of the paper-based chip:
s11, preparing a color development liquid: weighing 0.096 g of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) powder, dissolving in 1 mL of DMSO to prepare TMB mother liquor with the concentration of 400 mM, and diluting to 60-100mM for later use;
s12, preparing a horseradish peroxidase (HRP) mother solution: HRP was dissolved in phosphate buffer (PBS, 20 mM, pH = 7.4) to make 5000U/mL of stock solution. Continuously diluting to 20-100U/mL for later use;
s13, preparing a modifying liquid M: mixing a chitosan oligosaccharide lactic acid (COL) solution, a developing solution and a 25U/mL HRP solution according to the ratio of N: 1: 1 (volume ratio, N = 3-8) and uniformly mixing; chitosan oligosaccharide lactic acid (COL) solution is prepared by weighing 0.2-0.3 g COL powder (5-10 kDa), adding 9.8 mL water and 0.2 mL acetic acid, stirring and heating at 50 deg.C for 10min until chitosan oligosaccharide lactic acid is dissolved.
S2, preparing a required modification liquid N of the paper-based chip:
s21, preparing a Xanthine Oxidase (XOD) solution: XOD is dissolved in phosphate buffer (PBS, 20 mM, pH = 7.4) to prepare 100-500U/mL enzyme solution;
s22, preparing a modifying liquid N: uniformly mixing 300U/mL XOD solution and 1wt% chitosan solution according to an equal volume ratio; the 1wt% chitosan solution is prepared by weighing 0.05-0.1 g chitosan solution powder (100 kDa) and adding 9.8 mL water and 0.2 mL acetic acid, stirring and heating at 50 deg.C for 10min until the chitosan is dissolved.
S3, paper base cutting: cutting the nitrocellulose NC film into a square of 1.5 cm by 1.5 cm to be used as a paper base of the chip;
s4, modification in the first step: sucking 0.7 μ L of modifying solution M, dripping onto NC membrane, and drying at room temperature for 2 min; s5, second-step modification: after drying, 1 mu L of modification liquid N is dripped on the site which is just modified, and drying is carried out for 1.5 min at room temperature, thus finishing modification;
s6, according to the same method of S3-S4, at most 4 detection sites can be modified on a single paper base; the paper base modification method of the subsequent experiment was carried out according to this procedure without specific description.
S7, pre-packaging of the paper-based chip: packaging 5 manufactured paper-based chips into 6 cm by 10 cm aluminum foil vacuum packaging bags, and performing vacuum-pumping treatment and hot plastic packaging by using a vacuum-pumping machine;
s8, storage: and (4) storing the packaged paper-based chip in a refrigerator at 4 ℃ for later use.
A detection method for detecting a paper-based chip based on microfluid aggregated fish freshness specifically comprises the following steps:
t1, drawing a hypoxanthine standard curve:
t11, according to the optimized conditions, the method is applied to the detection of hypoxanthine. The concentration gradient of hypoxanthine is set to 0.01-5.2 mM;
t12, shooting the result, wherein the shooting specification is as follows: selecting a mobile phone photographing mode as a professional mode; the light source is selected as a built-in LED light source of the mobile phone; a sensitivity ISO selection of 50; selecting a light measuring mode as a spot light measuring mode; shutter speed S is set to 1/36; the exposure compensation EV is set to 0; the focusing mode AF is selected as single automatic focusing; the white balance mode WB is set to automatic white balance AWB; when the mobile phone takes a picture, the distance between the mobile phone and the target is 25 cm, and no external light source is arranged; and (3) color intensity modeling treatment: firstly, a picture shot by the mobile phone is transmitted to a computer and opened by using Image J software. And selecting 30 × 30 pixel units in a frame of the part with changed color in the picture by using a selection function, and obtaining the average R, G and B values of the selected area by using a software Histogram function Analyze-Histogram. Performing Euclidean distance model D processing on the obtained RGB value, wherein the Euclidean distance model D is represented by a formula (1); the results for color intensity in subsequent experiments were all processed in this way without special mention.
In the formula: r 0 ,G 0 ,B 0 Average RGB values of the blank group detection areas respectively; r i ,G i ,B i Respectively representing the average RGB values of the ith group of experimental color development areas;
drawing the result into a standard curve, and calculating a detection limit and a linear range;
t2, skin detection experiment of fish samples: taking out the sea bass blocks which are placed at 4 ℃ for 6, 5, 4, 3, 2 and 1 days, placing at room temperature for 10min, and then attaching the paper-based chip to the surface of the fish for 3 min, wherein the image acquisition and data processing method is T12;
t3, creation of mobile terminal freshness detection applet: in order to make the result more accurate and convenient when this freshness detection paper base chip carries out colorimetric estimation, we have tried to develop the removal end freshness detection applet. The mobile phone is used as an indispensable tool in daily life of people, and provides powerful support for field detection, wherein the small program function in the WeChat is convenient and quick and is often used by people in daily life. The acquisition of target color is realized by developing a small program function, a color signal is converted into an average RGB value, the numerical value is converted through a formula (1) and corresponds to a standard curve, the freshness of a sample is judged according to the obtained hypoxanthine concentration, and finally, an edible suggestion is given.
Example 2
The paper base material in step S3 was Whatman No. 1 chromatography paper, and the rest of the procedure was the same as in example 1.
Example 3
The paper base material in step S3 was baowerd filter paper made in china, and the rest of the steps were the same as in example 1.
Example 4
The paper base material in step S3 was dyed paper, and the rest of the procedure was the same as in example 1.
Preferably, the linear range of the detection of the target hypoxanthine in the step T12 is 0.01 mM-0.16 mM, the linear regression equation is y =492.722x + 32.798, wherein y is the color intensity, x is the concentration of the hypoxanthine, and the coefficient of determination R is 2 0.9903, the detection limit LOD is 8.22. mu.M. Therefore, the paper-based biosensor can be used for detecting the target hypoxanthine, thereby realizing the judgment of the freshness of the sample.
Color aggregation effect of paper-based chips
The color focusing effect of the modification method was studied, including the nitrocellulose membranes of examples 1-4, Whatman No. 1 chromatography paper, dyed paper, baowei germany filter paper, and the paper base was modified according to the same method, and as shown in fig. 2, only the nitrocellulose membrane exhibited a good color focusing performance, but other paper base materials had problems of insignificant color development, color dispersion, and the like. The reason is that the pore size of the NC membrane is 0.22 μm, which is much smaller than the pore sizes of the other three materials, so that the NC membrane can be better modified, and finally, the NC membrane presents better color aggregation.
Standard curve
The prepared nitrocellulose NC membrane paper-based chip is used for detecting the target hypoxanthine with the concentration range of 0.01 mM-5.2 mM, and water is used as a blank control. The experimental results are shown in fig. 3, the color of the nitrocellulose membrane paper-based chip gradually deepens and reaches the maximum within 3 minutes, and the preliminary signal interpretation can be easily carried out by naked eyes. The color signal is obtained by using a Huashi Mate 20 mobile phone, and the average RGB value of the color development area of the photographed part is read by using Image J software, so that the color intensity is increased along with the increase of the concentration of hypoxanthine; when the concentration of hypoxanthine reached 1.3 mM, the color intensity increased gradually and gradually. Wherein, the standard curve has better linearity when the concentration of hypoxanthine is 0.01-0.16 mM, and R is 2 0.9903 with a calculated detection limit of 8.22. mu.M, with a lower detection limit.
The linear range of the method for detecting the target hypoxanthine is 0.01 mM-0.16 mM, the linear regression equation is y =492.722x + 32.798 (y is color intensity, and x is the concentration of the hypoxanthine), and a coefficient (R) is determined 2 ) 0.9903, the limit of detection (LOD) is 8.22 mu M, so the paper-based biosensor can be used for detecting the target hypoxanthine, thereby realizing the judgment of the freshness of the sample.
Skin-on-skin detection of fish samples
The paper-based biosensor developed by the method has the great advantage that the paper-based biosensor can be directly attached to the surface of fish meat for testing the freshness without performing a pretreatment process on a fish meat sample. Therefore, the detection speed is greatly improved, the freshness detection is more convenient, and support is provided for the field detection of the freshness of aquatic products. As the fish meat is gradually rotten to produce hypoxanthine, the hypoxanthine on the surface of the fish meat is gradually accumulated along with the lapse of time. And the fish surface is comparatively moist environment, provides the prerequisite for the paper base chip can directly paste on the fish surface and detect.
The paper-based biosensor is respectively attached to the surfaces of the sea bass fishes placed at different times, whether a paper-based chip generates color signals or not is observed, and the generated color signals are acquired by using a Huashi Mate 20 mobile phone. The result is shown in fig. 4, the color of the paper-based chip is lighter on the first day of the sea bass preservation, which shows that the hypoxanthine accumulation is less and the fish flesh freshness is higher; the color of the paper-based chip begins to deepen on the second day and the third day of the sea bass preservation, which shows that the accumulation amount of hypoxanthine gradually increases and the freshness of fish gradually decreases from the second day; on the fifth and sixth days of the fourth day, the color displayed by the paper-based chip became very dark, indicating that the accumulation of hypoxanthine was large and that the fish meat had started to spoil.
Development of small program for quickly detecting freshness of mobile terminal
A WeChat small program of a mobile terminal is developed, the on-line freshness of aquatic products is evaluated, and corresponding eating suggestions are given.
The main functional area of the WeChat applet is divided into three areas, as shown in FIG. 5, the first area is an acquisition area of a color image, and information is uploaded to the color result of the paper-based biosensor through the photographing function of the smart phone; the second area is a selection module of a target color area, the module can select a color display part in the uploaded image, and an average RGB value in the area is read through a program; the third area is a blank control selection area, and can upload the area image of the blank test in the paper-based chip and read the average RGB value. And finally, processing and converting data through a background program, outputting a target area color result, and giving an edible suggestion.
In the description of the present specification, the description of "one embodiment," "some embodiments," "specific embodiments," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A manufacturing method of a paper-based chip for detecting freshness of fish meat based on microfluid aggregation is characterized by comprising the following steps:
s1, preparing a required modification liquid M of the paper-based chip:
s11, preparing a color development liquid: weighing 0.096 g of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) powder, dissolving in 1 mL of DMSO to prepare TMB mother liquor with the concentration of 400 mM, and diluting to 60-100mM for later use;
s12, preparing a horseradish peroxidase (HRP) mother solution: dissolving HRP in Phosphate Buffered Saline (PBS), 20 mM, and pH =7.4, preparing a mother solution with the concentration of 5000U/mL, and continuously diluting to 20-100U/mL for later use;
s13, preparing a modifying liquid M: mixing a chitosan oligosaccharide lactic acid (COL) solution, a developing solution and a 25U/mL HRP solution according to the ratio of N: 1: 1, and N =3-8, and uniformly mixing;
s2, preparing a required modification liquid N of the paper-based chip:
s21, preparing a Xanthine Oxidase (XOD) solution: dissolving XOD in phosphate buffer PBS (20 mM), and preparing an enzyme solution with the concentration of 100 and 500U/mL, wherein the pH is = 7.4;
s22, preparing a modifying liquid N: uniformly mixing a 300U/mL XOD solution with a 1wt% chitosan solution according to an equal volume ratio;
s3, paper base cutting: cutting the paper base material into squares of 1.5 cm by 1.5 cm to be used as paper bases of the chips;
s4, modification in the first step: 0.7 mu L of modification liquid M is absorbed and dripped on the cut paper base, and the paper base is dried for 2 min at room temperature;
s5, second-step modification: after drying, 1 mu L of modification liquid N is dripped on the site which is just modified, and drying is carried out for 1.5 min at room temperature, thus finishing modification;
s6, according to the same method of S3-S4, at most 4 detection sites can be modified on a single paper substrate;
s7, pre-packaging of the paper-based chip: packaging 5 manufactured paper-based chips into 6 cm by 10 cm aluminum foil vacuum packaging bags, and performing vacuum-pumping treatment and hot plastic packaging by using a vacuum-pumping machine;
s8, storage: and (4) storing the packaged paper-based chip in a refrigerator at 4 ℃ for later use.
2. The method for manufacturing the paper-based chip for detecting freshness of fish meat based on microfluidic aggregation according to claim 1, wherein the chitosan oligosaccharide lactic acid (COL) solution in step S13 is prepared by weighing 0.2-0.3 g of COL powder of 5-10 kDa, adding 9.8 mL of water and 0.2 mL of acetic acid, stirring and heating at 50 ℃ for 10min until the chitosan oligosaccharide lactic acid is dissolved.
3. The method for preparing a paper-based chip for detecting freshness of fish meat based on microfluid aggregation as claimed in claim 1, wherein the 1wt% chitosan solution in step S22 is prepared by weighing 0.05-0.1 g of chitosan solution powder 100-300 kDa, adding 9.8 mL of water and 0.2 mL of acetic acid, stirring and heating at 50 ℃ for 10min until chitosan is dissolved.
4. The method for making the microfluid aggregate fish flesh freshness detection paper-based chip according to claim 1, wherein the paper-based material in the step S3 comprises one of nitrocellulose membrane, Whatman No. 1 chromatography paper, baowerd filter paper made in China and dyed paper.
5. The manufacturing method of the microfluid-based aggregated fish flesh freshness detection paper-based chip according to claim 4, wherein the paper-based material is a Nitrocellulose (NC) film.
6. A detection method for detecting a paper-based chip based on microfluid aggregated fish freshness is characterized by comprising the following steps:
t1, drawing a hypoxanthine standard curve:
t11, hypoxanthine concentration gradient set to 0.01-5.2 mM;
t12, shooting the result, wherein the shooting specification is as follows: selecting a mobile phone photographing mode as a professional mode; the light source is selected as a built-in LED light source of the mobile phone; a sensitivity ISO selection of 50; selecting a light measuring mode as a spot light measuring mode; shutter speed S is set to 1/36; the exposure compensation EV is set to 0; the focusing mode AF is selected as single automatic focusing; the white balance mode WB is set to automatic white balance AWB; when the mobile phone takes a picture, the distance between the mobile phone and the target is 25 cm, and no external light source is arranged; and (3) color intensity modeling treatment: firstly, a picture shot by a mobile phone is transmitted to a computer and opened by using Image J software; selecting 30 × 30 pixel units from a frame of a part with changed colors in a picture by using a selection function, obtaining average R, G and B values of a selected region by using a software Histogram function Analyze-Histogram, and performing Euclidean distance model D processing on the obtained RGB values, wherein the Euclidean distance model D processing is shown in a formula (1);
in the formula: r 0 ,G 0 ,B 0 Average RGB values of the blank group detection areas respectively; r i ,G i ,B i Respectively representing the average RGB values of the ith group of experimental color development areas;
drawing the result into a standard curve, and calculating a detection limit and a linear range;
t2, skin detection experiment of fish samples: taking out the sea bass blocks which are placed at 4 ℃ for 6, 5, 4, 3, 2 and 1 days, placing at room temperature for 10min, and then attaching the paper-based chip to the surface of the fish for 3 min, wherein the image acquisition and data processing method is T12;
t3, creation of mobile terminal freshness detection applet: the acquisition of target color is realized by developing a small program function, a color signal is converted into an average RGB value, the numerical value is converted through a formula (1) and corresponds to a standard curve, the freshness of a sample is judged according to the obtained hypoxanthine concentration, and finally, an edible suggestion is given.
7. The method for detecting the paper-based chip based on the microfluid aggregated fish freshness detection, according to claim 6, wherein the linear range of the detection of the target hypoxanthine in the step T12 is 0.01 mM-0.16 mM, the linear regression equation is y =492.722x + 32.798, wherein y is color intensity, x is the concentration of the hypoxanthine, and the coefficient of determination R of the linear regression equation is 2 0.9903, the detection limit LOD is 8.22. mu.M.
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Citations (4)
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| US4105800A (en) * | 1976-07-26 | 1978-08-08 | Board Of Regents For Education Of The State Of Rhode Island | Immobilized enzyme method to assess fish quality |
| CN106610380A (en) * | 2015-10-26 | 2017-05-03 | 华东理工大学 | Method used for rapid detection of xanthine |
| CN109856102A (en) * | 2019-03-13 | 2019-06-07 | 福州大学 | A kind of hypoxanthic biosensor of detection aquatic products |
| CN114113506A (en) * | 2021-12-20 | 2022-03-01 | 合肥工业大学 | Method for judging freshness of meat by quantitatively detecting hypoxanthine |
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| NZ597217A (en) * | 2009-06-30 | 2013-02-22 | Univ Monash | Quantitative and self-calibrating chemical analysis using paper-based microfluidic systems |
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| US4105800A (en) * | 1976-07-26 | 1978-08-08 | Board Of Regents For Education Of The State Of Rhode Island | Immobilized enzyme method to assess fish quality |
| CN106610380A (en) * | 2015-10-26 | 2017-05-03 | 华东理工大学 | Method used for rapid detection of xanthine |
| CN109856102A (en) * | 2019-03-13 | 2019-06-07 | 福州大学 | A kind of hypoxanthic biosensor of detection aquatic products |
| CN114113506A (en) * | 2021-12-20 | 2022-03-01 | 合肥工业大学 | Method for judging freshness of meat by quantitatively detecting hypoxanthine |
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